I just changed target DNA solution from DMSO to arrayit micro spotting solution 2X plus, and after washing(N2 gas dry) I stacked my slides, but it spreaded so I cannot see dots..
spotting is Okay and no spresded after baking, so I think some problem occurs after washing(after pre hybridization).. what's the problem...?
P.S : where you solve oligonucleotide probe? I solve in 50% DMSO.
Is that Okay arrayit micro spotting solution 2X meets DMSO?
Did you scan the array from the right site?
Spotting
I have only bad experience with the spotting. I tried it once and decided to use commercial arrays. We used customized microarrays (8x15k or 8x60k) printed by Agilent Technologies.
But if you follow the ArrayIt instructions for spotting it should be OK.
Labeling
What kind of labeling system did you use? We labelled the DNA within an PCR reaction with Cy3 and Cy5-dUTPs and purified the reaction product with AmpureBeads?
My suggestion for the hybridization.
We used Agilent Oligo aCGH/Chip-on-chip Hybridization kit (Agilent Technologies) and followed the manufacturer’s instructions of the Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol (Version 6.0 November 2008; Agilent Technologies).
The hybridization was performed in an oven (65°C, 9 rpm) for 15 hours with protection from light to prevent degradation of the fluorescent dyes.
Thereafter, the array was washed three times with Wash Buffer 1 at room temperature for 5 minutes, followed by a final wash with Wash Buffer 2 for 30 min at 37°C.
After the final wash the array was dried at room temperature protected from dust in an open 50 ml conical tube.
Regards
Björn
@Björn Abendroth thank you for answer!
1. I use oligonucleotide probe, so i ordered cy3 labeled oligo. So I don't label for my oligo.
2. It was solved when I use wet kimtech(wet by DW), and do not stacks.. during hybridization, water can enter between slide and coverslip?
3. I spot oligos by hand, and use arrayit superamine2. spotting is okay except after baking, it remains white donught..